Biochemical and molecular mechanisms underlying the chemopreventive efficacy of rosmarinic acid in a rat colon cancer

European Journal of Pharmacology 791 (2016) 37–50

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Pulmonary, gastrointestinal and urogenital pharmacology

Biochemical and molecular mechanisms underlying the chemopreventive efficacy of rosmarinic acid in a rat colon cancer Karthikkumar Venkatachalam, Sivagami Gunasekaran, Nalini Namasivayam n Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar 608002, Tamilnadu, India

art ic l e i nf o

a b s t r a c t

Article history: Received 31 July 2015 Received in revised form 27 July 2016 Accepted 28 July 2016

To shed light on colon cancer chemoprevention, natural phytochemicals attract researchers by virtue of their beneficial biological effects. The chemopreventive potential of rosmarinic acid (RA) was tested by using the colon carcinogen, 1,2-dimethylhydrazine (DMH) by evaluating the Aberrant crypt foci (ACF), tumour incidence, lipid peroxidative byproducts, phase I & II drug metabolizing enzymes, cell proliferative and apoptotic proteins. Rats were divided into six groups and received modified pellet diet. Group 1 served as control rats, group 2 rats received RA (5 mg/kg b.w. p.o.), rats in groups 3–6 received DMH (20 mg/kg b.w., s.c.) for the first fifteen weeks. In addition to DMH, groups 4–6 received RA at the dose of 5 mg/kg b.w. during initiation, post initiation stages and also for the entire study period. DMH treated rats showed an increase in the development of ACF, tumour formation and multiplicity and decrease in lipid peroxidative byproducts. Moreover, it modulates xenobiotic enzymes and reduces the expressions of proapoptotic proteins; increases expressions of anti apoptotic proteins at the end of the study. Supplementation with RA to carcinogen treated rats protected them from the above deleterious effects caused by DMH and thus RA may be used as a potent chemopreventive agent. & 2016 Elsevier B.V. All rights reserved.

Keywords: DMH Apoptosis Antioxidants ACF Chemoprevention

1. Introduction Massive economic growth and rapid urbanization has influenced people to adopt unbalanced dietary style. It composed of high fat, high protein, low carbohydrate and low fiber which are considered to be the risk factors for colon cancer. Several epidemiological studies suggest that a positive correlation exists between decreased consumption of fruits and vegetables enriched in phytochemicals and the occurrence of colon cancer (van Duijnhoven et al., 2009). DMH is a procarcinogen that can induce colorectal adenocarcinoma in rats with high incidence and great specificity, especially when administered subcutaneously (Nalini et al., 1997). Aberrant crypt foci (ACF) are unique preneoplastic markers of colon cancer used to assess the chemopreventive effect of either a natural or synthetic compound. ACF can be classified according to their morphological subtypes and are identified by the (i) number of crypts (ii) crypt size and (iii) crypt multiplicity. Molan et al. (2014) reported that consumption of plant antioxidants promote the growth of beneficial bacteria and lower the numbers of other bacteria. β-glucosidase contributes to the hydrolysis of glucose monomers from nonstarch polysaccharides, n

Corresponding author. E-mail address: [email protected] (N. Namasivayam).

http://dx.doi.org/10.1016/j.ejphar.2016.07.051 0014-2999/& 2016 Elsevier B.V. All rights reserved.

however, it is also possible for β-glucosidase to be involved in the formation of toxic aglycons from plant glucosides (Pool-Zobel et al., 2002). Mucinase cleaves the protective mucin layer of the intestine. The change in mucin levels is an indicator for early malignant transformation. Another one enzyme which is important for prevention of colon cancer is nitroreductase, responsible for reducing nitrocompounds to aromatic amines (Gillette et al., 1968). Evasion of apoptosis is one of the major hall marks of tumour development, which involves dysregulations of many oncogenes and tumour suppressor genes. Bax enhances the apoptosis by binding to the antiapoptotic protein Bcl- 2 and formation of ionpermeable pores in mitochondrial membranes. Caspase 3 activated by upstream caspases-8, -9, catalyses the specific cleavage of many key cellular proteins mediating different signalling pathways. The downstream caspases are largely responsible for cleavage of many other cellular proteins, leading to the morphological manifestations of apoptosis (Blanc et al., 2000). Moreover, inhibition of chronic nuclear factor κB (NF-κB) activity can, in many cases, slow the growth of these tumour cell lines or induce cell death. Rosmarinic acid (RA), is a naturally occurring polyphenol found in Rosmarinus officinalis, Origanum vulgare and several other plant species (Vattem et al., 2005). It exerts various beneficial biological effects such as antioxidant, anti colon cancer and neuroprotective effects (Furtado et al., 2015; Moon et al., 2010; Ono et al., 2012) .

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K. Venkatachalam et al. / European Journal of Pharmacology 791 (2016) 37–50

2. Materials and methods

then the colons were removed and washed thoroughly with 0.9% NaCl, cut open longitudinally and placed on strips of filter paper with their luminal surface open and exposed. Another strip of filter paper was placed on top of the luminal surface. The colons were then secured and fixed in a tray containing 10% buffered formalin overnight. The colons were then stained with 0.2% methylene blue in order to identify ACF using a microscope as described by Bird and Good (2000). The total number of ACF/rat was calculated from the sum of all ACF. To determine crypt multiplicity, the number of aberrant crypts in each focus was recorded.

2.1. Chemicals and animals care

2.5. Lipid peroxidation byproducts and antioxidant enzymes

Acrylamide, bovine serum albumin (BSA), bromophenol blue, 1ʹ-chloro-2,4-dinitrobenzene (CDNB), 1,2-dimethylhydrazine (DMH), ethidium bromide, 2-mercaptoethanol, 5,5ʹdithiobis-2-nitrobenzoic acid (DTNB), nicotinamide adenine dinucleotide phosphate (NADP), rosmarinic acid (RA), sodium dodecyl sulphate (SDS) and N,N,N′,N′-tetra methyl ethylene diamine (TEMED) were purchased from Sigma Chemical Co. (USA). All other chemicals used were of analytical grade and obtained from Hi-Media Laboratories, Mumbai. Antibodies for p53, cleaved caspase-3 and -9 were purchased from Santa Cruz Biotechnology, USA. Male Wistar rats weighing 140–160 g were obtained from the Central Animal House, Rajah Muthiah Medical College and Hospital (RMMCH), Annamalai University, Tamil Nadu, India and housed under standard environmental conditions (12 h light/dark cycle; 50% humidity; temperature 25 72 °C). The modified pellet diet (20% of fat (Karthik Kumar et al., 2009)) and water were fed ad libitum to all the rats in the experiment. The rats were cared for in compliance with the principles and guidelines of the Ethical Committee for Animal Care of Annamalai University in accordance with the Indian National Law on Animal Care [Reg. No. (160/1999/ CPCSEA/437)].

The concentration of lipid peroxidation byproducts such as thiobarbituric acid reactive substances (TBARS) (Jiang et al., 1992), lipid hydroperoxides (LOOH) (Rao and Recknagel, 1968) and conjugated dienes (CD) (Ohkawa et al., 1979) were determined using standard methods as denoted in parentheses. The values are expressed as mmoles/mg tissue.

Previous studies from our group revealed that rosmarinic acid (RA) effectively prevents the colon cancer in short term (16 weeks) dose dependent study (Karthikkumar et al., 2012). The present long term study (30 weeks) was designed to gain an insight into the ability of RA to modulate antioxidant enzyme activities, lipid peroxidation byproducts and the expressions of apoptotic markers in carcinogen exposed rats.

2.6. Preparation of cytosolic and microsomal fractions Cytosolic and microsomal fractions were prepared from separate tissues (liver and colonic mucosa) were homogenized in 0.25 M sucrose and centrifuged at 9000  g for 20 min. The supernatant fluid was collected, 0.2 mL (v/v) of 0.1 M CaCl2 in 0.25 M sucrose was added to each, and the samples were kept on ice for 30 min, centrifuged at 27,000  g for 20 min, which yielded clear cytosolic fractions that were promptly assayed for phase II enzymes. Microsomal pellets were washed twice by suspending in 7 mL of 10 mM Tris-HCl (pH 7.4) and 0.25 M sucrose, centrifuged at 9000  g for 20 min, which yielded microsomal fractions, that were promptly assayed for phase I enzymes. 2.7. Measurement of Phase I and Phase II enzyme activity

2.2. RA, carcinogen administration and experimental protocol RA was suspended in water just before administration, everyday orally at the dose of 5 mg/kg b.w. for 30 weeks. DMH was dissolved in 1.0 mM EDTA just prior to use and the pH was adjusted to 6.5 with 1 mM NaOH to ensure the stability of the chemical. Rats were randomly divided into six groups of 6 rats each. Group 1 rats were considered as control (Control), group 2 received RA (5 mg/kg b.w.) every day throughout the experimental period (ControlþRA). Groups 3–6 received DMH (20 mg/kg b.w.) once a week subcutaneously for the first 15 weeks (DMH). In addition to DMH, group 4 received RA, as in group 2 for the first 15 weeks (Initiation; DMHþRA (I)), group 5 rats received RA, as in group 2 starting one week after the last DMH injection and continued till the end of the experimental period (Post-initiation; DMHþRA (PI)), group 6 rats received RA, as in group 2, throughout the experimental period (Entire period; DMH þRA (EP)) of 30 weeks. 2.3. Measurement of tumour incidence After termination of the experiment all rats were euthanized, rat colons were removed and flushed with potassium phosphatebuffered saline (0.1 M, pH 7.2). Tumours were counted through visual macroscopic examination and later verified with histopathological examination.

The content of cytochrome P450 was measured by the method of (Omura and Sato, 1964). Carbonmonoxide (CO) adducts are formed by the reaction of reduced cytochrome P450 with CO. The absorbance was measured at 450 nm. Cytochrome b5 content was measured by the method of (Omura and Sato, 1964). Cytochrome b5 content was calculated by measuring the spectral difference between reduced and oxidized cytochrome b5. CYP2E1 activity was measured by the method of Watt et al. (1997), the absorbance was measured at 450 nm. NADPH-cytochrome P450 reductase was measured by the method of Omura and Takesue (1970). The assay measures the rate of oxidation of NADPH at 340 nm. NADH-cytochrome b5 reductase was assayed by the method of Mihara and Sato (1972). The rate of reduction of potassium ferricyanide by NADH was measured at 420 nm. The activity of DT-diaphorase was assayed by the method of Ernster et al. (1962). The reaction was initiated with NADPH as the electron donor and 2,6-dichlorophenol indophenol (DCPIP) as the electron acceptor. The reduction of DCPIP was measured spectrophotometrically at 600 nm. The activity of GST was assayed by the method of Habig and Jakoby (1981). GST activity was measured by following the increase in absorbance at 340 nm using 1-chloro2,4-dinitrobenzene (CDNB) as the substrate. UDPGT was assayed by the method of Isselbacher et al. (1962). The disappearance of p-nitrophenol upon glucuronidation was used to measure UDPGT activity. 2.8. Measurement of faecal and mucosal bacterial enzymes activity

2.4. Determination of ACF At the end of the experimental period, rats were killed and

Fresh faecal specimens were collected and suspended in icecold phosphate buffer (0.1 M, pH 7.0) and homogenized in a pre-

K. Venkatachalam et al. / European Journal of Pharmacology 791 (2016) 37–50

chilled homogeniser and the homogenate was then subjected to centrifugation at 2000  g for 10 min. The supernatant was used for the various enzyme assays. For mucosal samples, the rat colon was placed on an ice-cold polystyrene plate and cut open longitudinally. The mucosal layer was scraped gently using an ice-cold microscopic slide to collect the mucosa. The mucosal scrapings were homogenized in ice-cold phosphate buffer (0.1 M, pH 7.0) with a Teflon pestle in a glass tube, centrifuged at 12,000  g for 20 min at 4 °C and the supernatant was used for the assay of microbial enzymes. The measurements of faecal and mucosal bacterial enzymes were elaborated previously by our group (Karthik Kumar et al., 2009). The activity of β-glucuronidase was measured by the method of Freeman (1986). The amount of p-nitrophenol liberated (yellow colour) from the substrate p-nitrophenyl β-D-glucopyranoside in alkaline solution by the catalytic action of the enzyme β-glucuronidase was read in a spectrophotometer at 540 nm. The activity of β-glucosidase was measured by the method of Freeman (1986). The method is based on the spectrophotometric determination of yellow coloured p-nitrophenol liberated from the substrate p-nitrophenol β-D-glucoside by the action of β-glucosidase. The colour developed was read at 450 nm. The activity of β-galactosidase was measured by the method of Freeman (1986). p-nitrophenol liberated from the substrate p-nitrophenyl-β-D-galactopyranoside in alkaline solution by the action of β-galactosidase was measured spectrophotometrically at 405 nm. Mucinase activity was measured by the method of Shiau and Chang (1983). The amount of reducing sugar (glucose) released from mucin by the action of mucinase was measured spectrophotometrically at 610 nm. Nitroreductase activity was measured by the method of Bratton and Marshall (1939). The amount of paminobenzoic acid formed from p-nitrobenzoic acid in 70% ethanol by the action of the enzyme nitroreductase was read spectrophotometrically at 550 nm. Sulphatase activity was measured by the method of Rowland et al. (1983). The amount of p-nitrocatechol liberated from p-nitrocatechol sulphate in acetate buffer by the action of the enzyme sulphatase was read spectrophotometrically at 490 nm. 2.9. Histopathological analysis For histopathological examination, a portion of colonic tissue was fixed in 10% neutral buffered formalin routinely processed, embedded in paraffin and stained with haematoxylin and eosin (H&E). 2.10. PCR primer design Primers were designed using the primer express software (Eppendorf, USA). Primers specific for rat p53 (Gene of interest) and internal standard GAPDH were used. p53 Forward primer: 5′- AAA GGA TGC CCG TGC TGC CG -3′ Reverse primer: 5′- GCG GGA CGT AGA CTG GCC CT -3′ GADPH. Forward primer: 5′-CTG CAC CAC CAA CTG CTT AGC C-3′ Reverse primer: 5′-ACA GCC TTG GCA GCA CCA GT-3′ Real time PCR conditions and analysis. The real time PCR reactions were analysed in 96-well plates with a Realplex detection system (Eppendorf) using one step SYBR Green (QuantiFast SYBR Green RT-PCR Kit, QIAGEN, Germany). The total volume of 25 mL reaction mixture contains 200 nM of each primer, 5 mL total RNA and 12.5 mL 2  SYBR Green Master Mix Reagent. Reactions were run using the manufacturer's recommendations for first step cycling, reverse transcription (50 °C for 10 min) and PCR initial step (95 °C for 5 min), and two step

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cycling 30 cycles of 95 °C for 15 s (denaturation) and 60 °C for 1 min (combined annealing/extension). The expression levels were calculated from the number of amplification cycles needed to reach a fixed threshold in the exponential phase of the PCR reaction (Ct). Data were analysed using the Realplex cycler (Eppendorf). Once the calculation for each gene was performed, the results were normalized to the GAPDH reference gene. 2.11. Immunoblot analysis To collect the protein samples, colonic mucosal cells were gently scraped off using a glass slide, then the scrapings were homogenized in protein buffer (10% Tris-HCl, 5% sucrose, 1% EDTA, 1% EGTA, 0.0125% NaF, 10% Triton X-100, 1% sodium orthovanadate, 4% Sigma protease inhibitor cocktail, 0.07% β-mercaptoethanol) and the mucosal cells were lysed and homogenized in a tissue homogeniser. The lysates of mucosal cells were centrifuged at 15,000  g for 20 min at 4 °C, the supernatants were collected and the following parameters were evaluated. Each protein (50 mg) samples were separated through SDSPAGE. The separated proteins were electrophoretically transferred to polyvinylidene difluoride membranes. Protein blots were incubated with 1  PBS containing 5% non-fat dry milk for 2 h to block unspecific binding sites. The blot was incubated with 1:1000 dilution of primary antibody overnight at 4 °C. After washing, the blots were incubated with 1:2000 dilution of horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Following adequate washes, the immunoreactive proteins were visualized using enhanced chemiluminescence detection reagents (Sigma) and quantitated by Image J, a public domain Java image processing software, Wayne Rasband, NIH, Bethesda, MD, USA. βactin was used as the house keeping loading control. 2.12. Immunohistochemical analysis Formalin fixed, paraffin embedded sections of colon tissue from animals were assayed for various proteins by immunohistochemical staining. Briefly, deparaffinized sections were blocked for endogenous peroxidase activity with H2O2 (0.3% in PBS). Antigen retrieval was performed by fractioned microwave treatment in citrate buffer (pH 6.0). Subsequently, sections were covered for 30 min at room temperature with 0.04% casein to block the non-specific proteins, followed by incubation with monoclonal antibody for 30 min at 37 °C. Later, slides were covered with appropriate biotinylated secondary antibody at room temperature and streptavidin biotin horseradish peroxidase complex (Nova castra) for 30 min. Sections were then developed using diaminobenzidine as the chromogen. The slides were counterstained with haematoxylin. 2.13. Statistical analysis Values are given as the means 7 S.D. Significant difference between the means of the six groups was statistically analysed using one way analysis of variance (ANOVA) and Duncan's Multiple Range Test (DMRT). The significance levels was set at Po 0.05 for all the tests. Statistical analysis was performed using SPSS 11.0 software package (SPSS, Tokyo, Japan).

3. Results During the study, no clinical signs of toxicity were present in any group. Histologically there were no pathological alterations indicative of RA toxicity in the major organs.

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K. Venkatachalam et al. / European Journal of Pharmacology 791 (2016) 37–50

Table 1 Effect of DMH and RA on body weight changes. Groups

Initial body Final body Weight gain weight (in gm) weight (in gm) (in gm)

Control Controlþ RA (5 mg/kg b.w.) DMH DMH þRA (Initiation) DMH þRA (Post initiation) DMH þRA (Entire period)

155.0 7 13.4 153.0 7 14.2 154.8 7 12.4 152.5 7 14.8 157.4 7 14.6 156.8 7 12.8

287.6 7 26.6a 275.4 7 25.5a 216.2 7 19.8c 242.7 7 22.5b 244.8 7 22.7b 264.27 24.5ab

132.6 7 12.3a 122.4 7 11.4a 61.4 7 5.7d 90.2 78.4c 87.4 7 8.1c 107.4 7 10.0b

Values are means 7 SD of each group (n¼ 12). Groups not sharing a common superscript letter (a-d) differ significantly at P o 0.05 (DMRT).

3.1. Body weight changes Data presented in Table 1 shows the body weight changes and weight gain of experimental rats. The weight gained by rats in the control 4controlþRA DMH þRA (Entire period) 4DMHþRA (post-initiation) 4DMH þRA (initiation) 4 DMH. The final body weight of DMH alone treated rats (group 3) were significantly lower than that of the control rats (group 1). Weight gain was calculated as the difference between the final and the initial body weight of the rats. RA supplemented DMH treated rats (groups 4, 5, and 6) showed a significant (P o0.05) increase in the growth rate as compared to the unsupplemented DMH treated rats (group 3). 3.2. The incidence of tumour No tumours were observed in the control and control þRA treated rats (groups 1 and 2). Carcinogen exposed rats shows 100% incidence and total number of tumour. Whereas, carcinogen exposed RA treated rats shows the reduced tumour incidence and total number of tumour, it shows the tumour inhibiting potential of RA (Table 2, Fig. 1). 3.3. ACF and DACF counting All the rats belonging to the groups 3–6 on exposure to DMH developed ACF. In group 3, DMH induced rats showed increased total number of ACF and multiplicity. The frequency and multiplicity of ACF/colon, on supplementation with RA to DMH treated rats (groups 4–6) was significantly (p o0.05) lower as compared to the DMH alone treated rats (group 3). These results showed that RA significantly inhibited DMH-induced ACF formation and multiplicity, the effect was more pronounced when RA was supplemented for the entire study period (group 6). Percentage

Fig. 1. Macroscopical observation of the rat colon.

inhibition of ACF in RA supplemented rats was 34.61%, 28.84%, and 71.15% in groups 4, 5, and 6 respectively (Table 2, Fig. 2). 3.4. The levels of lipid peroxidation byproducts A significant increase (Po 0.05) in the levels of LOOH and CD was observed in the liver and significantly decreased levels were observed in the colon of DMH alone treated group (group 3) (Table 3) as compared to the control rats (group 1). At the same time the LOOH and CD levels were decreased in DMH alone treated rats. Supplementation with RA to DMH treated rats during the initiation, post-initiation and entire period stages (groups 4–6) significantly restored the levels of LOOH and CD of the liver and colon to near those of control rats. 3.5. Effect of DMH and RA on xenobiotic metabolizing enzymes The activities of the CYP 450, CYP2E1 and NADPH-CYP450 reductase enzymes (Table 4) in the liver and colon microsomes were increased significantly on exposure to DMH (group 3). These

Table 2 Effect of RA on DMH induced ACF, DACF formation and tumour incidence. Groups

Number of ACF per colon 1 crypt

DMH DMH þRA (Initiation) DMH þRA (Post initiation) DMH þRA (Entire period)

2 crypts

3 crypts

Total

% Inhibition DACF

4 or more crypts

147 1.24a 187 1.60a 11 70.98a 9 70.80 117 0.98b 127 1.06b 77 0.62b 4 70.35

a b

527 4.61a – 347 3.02b 34.61

3 70.28 1 70.08

Tumour inNo. of tumour bearing cidence (%) rats (No. of rats examined)

Total tumour number

No. of tumours/ tumour-bearing rat

6 (6) 2 (6)

100 33

10 3

1.67 1.50

97 0.80c

147 1.24c

97 0.80c

5 70.44

c

377 3.28c

28.84

1 70.084 4 (6)

67

6

1.50

57 0.44d

7 70.62d

17 0.09d

2 70.18

d

15 71.33d

71.15

–

11

1

1.00

1 (6)

Values are means 7SD of each group (ACF: n ¼ 6, Tumour incidence: n ¼12). Groups not sharing a common superscript letter (a-d) differ significantly at P o 0.05 (DMRT).

Tumour incidence( %) =

No . of tumour bearing rats No . of rats examined

× 100

K. Venkatachalam et al. / European Journal of Pharmacology 791 (2016) 37–50

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Fig. 2. Topographical view of normal and aberrant crypts (arrows) in the colonic mucosa of experimental rats stained with methylene blue (n ¼6). A and B: Control and Controlþ RA rat colon shows normal crypts; C: DMH rat colon shows ACF having more than eight crypts; D and E: DMH þRA (Initiation & Post initiation) rat colon shows five aberrant crypts; F: DMH þ RA (Entire period) rat colon shows two crypts; G and H: Dysplastic ACF of DMH and DMH þRA (Entire period) rat colon respectively.

deleterious effects were reversed to near normal values on supplementation with RA to the DMH exposed rats (groups 4–6). Table 5 represents the activities of NADH-cytochrome b5 reductase and cytochrome b5 in the liver and colon microsomes of experimental rats. The increased levels of these enzymes were observed in carcinogen only exposed rats (group 3). Whereas, RA supplementation to DMH treated groups (groups 4–6) sturdily reduced the levels of these enzymes. However, RA alone treated rats (group 2) did not show any significant change as compared to the control rats (group 1).

Decreased activities of phase II enzymes were observed in DMH alone treated rats as compared to the control rats. On the other hand supplementation with RA to DMH injected rats during the different stages of carcinogenesis (group 4–6) significantly increased the activities of phase II enzymes activities as compared to the DMH alone treated rats (group 3). Thus supplementation with RA to DMH treated rats upregulated the activities of most of the phase II enzymes such as UDPGT, GST and DTD in the liver and colonic tissues (Table 6).

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Table 3 Effect of RA and DMH on tissue LOOH and CD.

LOOHa

CDb

Parameters

Control

ControlþRA

DMH

DMHþ RA (Initiation)

DMH þ RA (Post initiation)

DMHþ RA (Entire period)

Liver Proximal colon Distal colon Liver Proximal colon Distal colon

65.7 7 9.11a 73.7 7 7.1a 69.3 7 6.7a 63.0 7 5.9a 75.17 7.5a 77.6 7 7.4a

68.27 6.43ab 74.3 7 6.1b 72.3 7 7.0b 70.17 6.8ab 77.0 7 7.4a 81.3 7 8.0a

82.6 7 7.8c 49.2 74.8c 55.6 7 5.2c 90.8 7 9.1d 59.4 7 5.5c 62.6 7 6.2c

75.5 7 6.9b 58.17 5.4bc 58.2 7 5.0c 80.3 7 7.9c 67.3 7 6.2b 69.3 7 7.2d

69.2 76.7a 65.8 76.1a 65.3 76.4a 72.2 7 7.1b 61.3 7 6.0b 72.5 7 5.1bd

65.17 6.1aA 69.4 76.7aA 68.47 6.3aA 66.57 6.6aA 73.5 7 7.4aA 75.6 77.4bA

Values are means 7SD of each group (n¼ 6). Groups not sharing a common superscript letter (a-e) differ significantly at P o 0.05 (ANOVA followed by DMRT). a

mmoles/mL. mmoles/mL. A P o 0.001, Significantly different from DMH treated and RA supplemented groups (Groups 4–6). b

Table 4 Effect of RA on tissue CYP 450, CYP2E1 and NADPH-CYP450 reductase of experimental rats.

CYP450

CYP4502E1

NADPH-CYP450 reductase

Groups

Control

ControlþRA

DMH

DMH þRA (Initiation)

DMH þRA (Post initiation)

DMHþ RA (Entire period)

Liver Proximal Colon Distal Colon Liver Proximal Colon Distal Colon Liver Proximal Colon Distal Colon

4.137 0.3a 2.197 0.2a

4.157 0.4a 2.017 0.1a

7.95 7 0.76c 3.977 0.3c

6.88 7 0.6b 3.217 0.3b

5.81 7 0.5b 3.06 70.2b

4.747 0.4aA 2.2470.2aA

1.93 7 0.1a

1.98 7 0.1a

3.417 0.3c

2.57 70.2b

2.45 7 0.1b

1.81 7 0.1aA

5.43 7 0.5a 1.17 70.1a

5.047 0.4a 1.077 0.1a

9.48 7 0.91c 2.447 0.2d

7.75 7 0.7b 1.88 7 0.1c

7.39 70.7b 1.42 70.1b

5.86 7 0.5aA 1.22 7 0.1bA

0.95 7 0.09ab

0.917 0.08a

1.83 7 0.17d

1.42 7 0.1c

1.34 70.1c

1.08 7 0.1bA

63.2476.0a 11.22 7 1.0a

63.497 6.1a 10.357 0.9a

86.447 8.3c 27.337 2.6c

77.01 77.4b 21.93 72.1b

74.46 7 7.1b 18.107 1.7 b

67.83 7 6.5aA 12.75 7 1.2aA

10.55 71.0a

10.047 0.9a

23.40 72.2c

19.0771.8b

15.917 1.5b

11.88 7 1.1aA

Values are means 7SD of each group (n¼ 6). Groups not sharing a common superscript letter (a-e) differ significantly at P o 0.05 (ANOVA followed by DMRT). A

P o 0.001, Significantly different from DMH treated and RA supplemented groups (Groups 4–6).

Table 5 Effect of RA on tissue NADH-cytochrome b5 reductase and cytochrome b5 of control and experimental rats.

NADH-cyt b5 reductase

Cyt b5

Groups

Control

ControlþRA

DMH

DMH þRA (Initiation)

DMHþ RA (Post initiation)

DMHþ RA (Entire period)

Liver Proximal Colon Distal Colon Liver Proximal Colon Distal Colon

21.42 7 2.0a 5.96 7 0.5a

20.60 7 1.9a 6.197 0.6a

30.95 7 2.9c 16.32 7 1.5d

28.50 7 2.7b 13.05 7 1.2c

26.46 72.5b 10.81 7 1.0b

22.69 7 2.1aA 7.08 7 0.6bA

4.99 7 0.5ab

5.45 7 0.52a

13.97 71.3d

10.91 71.0c

8.36 70.8c

6.32 7 0.6bA

21.42 7 2.0a 5.96 7 0.5a

20.60 7 1.9a 6.197 0.5a

30.95 7 2.9d 16.32 7 1.5e

28.50 7 2.7c 13.05 7 1.2d

26.46 72.5b 10.81 7 1.0c

22.69 7 2.1aA 7.08 7 0.6bA

4.99 7 0.4a

5.45 7 0.5a

13.97 71.3c

10.91 71.0b

8.36 70.8b

6.32 7 0.6aA

Values are means 7SD of each group (n¼ 6). Groups not sharing a common superscript letter (a-e) differ significantly at P o 0.05 (ANOVA followed by DMRT). A

P o 0.001, Significantly different from DMH treated and RA supplemented groups (Groups 4–6).

3.6. Effect of DMH and RA on faecal and mucosal bacterial enzymes activity The activities of these enzymes were significantly elevated in the DMH alone treated rats (group 3) at the end of 30 weeks as compared to the control and RA treated control rats (groups 1 and 2). Supplementation with RA to DMH treated rats significantly (P o0.05) decreased their activities as compared to the DMH alone treated rats. RA supplementation during the initiation, post initiation and the entire period treatment regimens remarkably decreased the activities of the above enzymes that were

statistically significant (Po0.05) as compared to the unsupplemented DMH treated rats (Table 7). The activities of mucosal bacterial enzymes in DMH-treated rats (group 3) were significantly increased (P o0.05) as compared to the control group (group 1). RA supplementation during the initiation, post-initiation and entire period stages (group 4–6) of carcinogenesis significantly decreased the activities of mucosal bacterial enzymes as compared to DMH alone treated rats. This effect was more pronounced in the entire period RA treatment group (group 6) as compared to the other RA treated rats (Table 8).

K. Venkatachalam et al. / European Journal of Pharmacology 791 (2016) 37–50

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Table 6 Effect of RA on tissue UDPGT, GST and DTD of experimental rats.

UDPGT

GST

DTD

Groups

Control

ControlþRA

DMH

DMH þ RA (Initiation)

DMHþ RA (Post initiation)

DMHþ RA (Entire period)

Liver Proximal Colon Distal Colon Liver Proximal Colon Distal Colon Liver Proximal Colon Distal Colon

6.38 7 0.6a 3.46 7 0.3a

6.22 7 0.5a 3.577 0.3a

3.417 0.3c 1.42 7 0.1c

4.13 7 0.3b 1.98 7 0.1b

4.98 7 0.4b 2.65 7 0.2b

6.147 0.5aA 3.217 0.3aA

2.74 70.2a

2.62 7 0.2a

0.96 7 0.09c

1.737 0.1b

2.047 0.2b

2.55 7 0.2aA

1.83 7 0.1a 2.32 7 0.2a

1.78 7 0.1a 2.30 7 0.2a

0.95 7 0.09c 1.22 7 0.1d

1.30 7 0.1b 1.41 7 0.1c

1.617 0.1b 1.73 70.1b

1.73 70.1aA 2.09 7 0.2bA

1.88 7 0.1ab

1.8117 0.1a

0.91 70.08d

1.34 7 0.1c

1.377 0.1c

1.707 0.1bA

2.03 7 0.2a 1.60 7 0.1a

2.017 0.2a 1.517 0.1a

1.04 70.1d 0.82 7 0.08e

1.28 7 0.1c 1.05 7 0.1d

1.667 0.1b 1.217 0.1c

1.93 7 0.1aA 1.58 7 0.1bA

1.417 0.1a

1.44 70.1a

0.667 0.06c

0.84 70.08b

1.197 0.1b

1.34 7 0.1aA

Values are means 7SD of each group (n¼ 6). Groups not sharing a common superscript letter (a-e) differ significantly at P o 0.05 (ANOVA followed by DMRT). A

P o 0.001, Significantly different from DMH treated and RA supplemented groups (Groups 4–6).

Table 7 Effect of RA on faecal bacterial enzymes of control and experimental rats. Groups

Control

Controlþ RA

DMH

DMHþ RA (Initiation)

DMHþ RA (Post initiation)

DMlHþ RA (Entire period)

β-glucuronidaseA β –glucosidaseA β –galactosidaseA MucinaseB NitroreductaseD SulphataseC

32.13 73.0a 109.34 7 10.5a 28.05 7 2.7a 3.16 70.3a 20.247 1.9a 26.54 7 2.5a

33.66 73.2ab 117.81 711.3a 29.32 72.8a 3.21 70.3ab 21.42 7 2.0a 26.39 72.5a

53.80 75.1d 196.29 7 18.8d 64.577 6.2d 5.58 70.5e 35.547 3.4d 44.12 74.2d

43.75 7 4.2c 178.09 7 17.1c 55.39 7 5.4c 4.647 0.4d 29.89 7 2.8c 38.55 7 3.7c

38.40 73.6b 141.277 13.5ab 36.217 3.4b 4.02 70.3c 25.047 2.4b 33.63 7 3.2b

35.197 3.3ab 124.797 12.0ab 31.62 7 3.0a 3.6770.3bc 22.187 2.1ab 29.58 7 2.8a

Data are presented as the means 7 SD of each group (n¼ 6). (a-e) P o 0.05 the values not sharing a common superscript letter are significantly different from the DMH-treated groups (ANOVA followed by DMRT). A

mg of p-nitrophenol liberated/min/g protein. mg of glucose liberated/min/mg protein. C mmoles of p-nitrocatechol liberated/min/g protein. D mmoles of p-aminobenzoic liberated/min/g protein. B

Table 8 Effect of RA on mucosal bacterial enzymes of control and experimental rats. Groups

Control

Controlþ RA

DMH

DMH þRA (Initiation)

DMH þ RA (Post initiation)

DMlHþ RA (Entire period)

β-glucuronidaseA β –glucosidaseA β -galactosidaseA MucinaseB NitroreductaseC SulphataseD

10.717 1.0a 29.0772.7a 18.87 7 1.8a 6.73 70.6a 16.147 1.5a 20.22 7 1.9a

10.91 71.0a 31.417 3.0a 20.917 2.0a 6.98 70.6a 16.43 7 1.5a 22.047 2.1a

19.43 7 1.8d 61.65 7 5.9d 35.197 3.3d 12.2407 1.1d 31.69 73.0d 41.76 74.0d

17.34 7 1.66c 48.247 4.6c 29.32 7 7 2.8c 9.337 0.898c 27.28 7 2.6c 35.017 3.3c

14.487 1.3b 37.687 3.6ba 25.50 72.4b 8.2117 0.7b 23.007 2.2b 30.66 7 2.9b

11.83 7 1.1a 32.53 7 3.1a 21.93 7 2.1a 6.987 7 0.6a 18.56 7 1.7a 23.65 7 2.2a

Data are presented as the means 7 SD of each group (n¼ 6). a-e P o 0.05 the values not sharing a common superscript letter are significantly different from the DMH-treated groups (ANOVA followed by DMRT). A

mg of p-nitrophenol liberated/h/g protein. mg of glucose liberated/min/mg protein. mmoles of p-aminobenzoic liberated/min/g protein. D mmoles of p-nitrocatechol liberated/min/g protein. B C

3.7. Effect of DMH and RA on histopathological changes Figs. 3 and 4 shows the histopathological evidence of experimental groups. Carcinogen alone exposed rats (group 3) colon shows the lake of mucin in which tumour cells float and the tumour cells tend to form glandular units. RA supplementation to carcinogen exposed rats showing lymphoid aggregation and preneoplastic lesions (groups 4–6). DMH treated rats liver tissue shows anisocytosis, nuclear pleomorphism, occasional mitotic figures. Portal traid surrounded by inflammatory cell infiltrations

(group 3). RA supplemented DMH exposed rats minimize the deleterious effects; it shows only the fatty changes of micro and macro vesicular type. 3.8. mRNA expressions of p53 The relative values of the mRNA expression of p53 was represented in Fig. 5. p53 is a gatekeeper gene, which downregulated in the colonic mucosa of the DMH alone exposed rats (group 3) as compared to control rats (group 1). On supplementation with RA to

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Fig. 3. (A & B): 20  Colonic sections showing mucosa, submucosa and serosa within normal limits, (C) 40  Tumour cells ( ) tend to form glandular units, cystically dilated filled with mucin ( ), (D) 20  Tumour cells ( ) tend to form glandular units surrounded by scattered lymphocytes, (E) 40  Mucosa showing aberrant crypt foci, (F) 20  Lymphoid aggregates and submucosa showing aberrant crypt foci. (n ¼ 6).

DMH-exposed rats (groups 4–6), the expression of p53 was upregulated in the different treatment regimens as compared to unsupplemented DMH-treated rats (group 3). 3.9. Immunoblot expression levels of p53, caspase-3, -9 and p65 Fig. 6 illustrates the effect of RA on the expressions of p53, caspases 3, 9 and p65 in the colonic tissues of experimental rats. The mean colonic protein expression from control samples was designated as 100% in the graph. No significant changes in the expression of the proapoptotic proteins and p65 were observed in rats supplemented with RA alone (group 2) as compared to the control. Densitometric analysis of DMH treated colonic mucosa revealed significantly (P o0.05) lowered expressions of p53, caspase 3 and 9 whereas p65 significantly increased protein expression as compared to control (group 1). Supplementation with RA significantly elevated the expression of p53 and caspases and reduces the levels of p65 in all the three treatment groups. 3.10. Immunohistochemistry of Bcl-2, Bax and COX-2 The effect of RA on immunolocalisation of Bcl-2, Bax and COX-2 in the colonic mucosa of experimental animals are shown in

Figs. 7–9. Pro- and anti apoptotic proteins and inflammatory proteins such as Bax, Bcl-2 and COX-2 were assayed from colorectal tissue sections of rats treated with DMH. Administration of DMH for 15 weeks significantly increased the Bcl-2 and COX-2 expressions and decreased Bax expression at the end of the experimental period of 30 weeks in DMH alone administered rats (group 3) as compared to the control rats (group 1). Supplementation with RA to DMH treated rats during the initiation (group 4), post initiation (group 5) and entire period treatment protocol (group 6) decreased Bcl-2 and COX-2; increased Bax expressions as compared to DMH alone treated rats (group 3). No significant changes in the expressions of Bcl-2, Bax and COX-2 were observed in the rats supplemented with RA alone (group 2) as compared to control rats (group 1).

4. Discussion The countries with high mortality rate with huge proportion of colorectal cancer patients may reflect the adoption of western lifestyle, food behaviour and physical inactivity (Center et al., 2009). Dietary habits are playing significant role in development of colon cancer. A diet containing high fat, high carbohydrate, low

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Fig. 4. Liver sections showing central vein traid and portal. A and B: Control, (A, B) 20 - Liver showing central vein; sinusoidal dilation, (C) (20  ) Hepatocytes showing anisocytosis, nuclear pleomorphism, occasional mitotic figures. Mono nuclear giant cell transformation. Portal traid surrounded by inflammatory cell infiltrations. (D) (20  ) Showing fatty change. There is anisocytosis of cells with nuclear pleomorphism. Occasionally blastic transformation of hepatocytes noticed, (E, F) (20  ) Hepatocytes shows fatty change of micro & macro vesicular type. (n ¼ 6).

Fig. 5. mRNA expression of p53 in the colon of experimental rats. Values are means 7 SD of each group (n ¼ 6). Groups not sharing a common superscript letter (a–c) differ significantly at po 0.05 (DMRT).

fiber is more vulnerable than the low fat diet. Prevention of cancer with the use of dietary molecules may pave the way to eradicate the disease.

In our study, DMH was used as an inducer and peanut oil as a promoter to accelerate colon carcinogenesis in rats. The increased incidence of aberrant crypts in the DMH exposed rats could have been enhanced by the presence of peanut oil as a tumour promoter. We noticed a significant (P o0.05) decrease of weight gain and growth rate in carcinogen exposed rats (groups 3–6), it may be due to altered metabolism, tumour burden of the colon cancer bearing rats (Fig. 1), which supports our findings that the occurrence of tumour is more prevailed in DMH alone exposed rats shows that collapse of protective strategies, followed by the RA supplemented rats (group 3 4group 5 4group 4 4group 6) suggesting less aggressive behaviour of the tumour in the presence of RA. In this context it is known that high energy levels are required by cancer cells for their relentless proliferation. On the other hand, supplementation of RA prevents the drastic reduction of weight loss in DMH exposed rats shows its efficacy against colon cancer. RA also reduces the tumour burden in carcinogen treated rats (67%, 33%, 89%) by its antitumour properties which emphasizes its huge benefit against DMH induced colon cancer. The increased incidence and multiplicity (i.e four or more crypts) of ACF in the DMH treated colon implies a high risk of colon cancer, as compared to the colon containing low incidence of

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Fig. 6. A . Immunoblot analysis of colonic p53, caspases -3, -9, and p65 expression. B. Each lane was analysed by densitometry and the expression in the control was considered as 100%. The column heights are the means 7 S.D. of six determinants (n ¼ 6). *Significantly (P o 0.05) different from control groups.

Fig. 7. Immunohistochemical expression of Bcl2 in the colon of control and experimental rats. A and B: Colonic sections of control and control þRA showing normal Bcl-2 expression. C: Colonic section of DMH treated rat showing intense Bcl-2 expression. D, E and F: Colonic sections of DMH treated rats supplemented with RA showing moderate to weak Bcl-2 expressions during the initiation, post initiation and entire period treatment regimens (n ¼6).

K. Venkatachalam et al. / European Journal of Pharmacology 791 (2016) 37–50

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Fig. 8. Immunohistochemical expression of BAX in the colon of control and experimental rats. A and B: Colonic sections of control and controlþ RA showing standard Bax expression. C: Colonic section of DMH treated rat showing weak Bax expression. D, E and F: Colonic sections of DMH treated rats supplemented with RA showing mild to moderate Bax expressions during the initiation, post initiation and entire period treatment regimens (n ¼6).

ACF and its multiplicity (Aranganathan et al., 2009; Wei et al., 2003). DMH induced ACF formation and multiplicity reflects the initiation stage of colon cancer in rats. In our study in addition to DMH, high fat diet also increased the multiplicity of ACF by its tumour promoting capabilities. Supplementation with RA (5 mg/ kg b.w.) to DMH exposed rats apparently reduced the formation of ACF and its replication. Besides, increased number of ACF, we have also observed luminal alterations, goblet cell reduction and nuclear alterations of the cells surrounding the lumen of the crypt. These characteristics can be correlated with increased multiplicity of ACFs. These results suggested that the growth of some ACFs (as assessed by increased ACF multiplicity) is accompanied by acquisition of dysplastic features of precancerous lesions. The number of foci consisting of one, two and greater than four dysplastic crypts were significantly lower in rats supplemented with RA and DMH than those of rats treated with DMH alone. In particular, supplementation with RA for the whole period of the experiment showed the greatest inhibitory potential on ACF formation and multiplicity. In this context, it is already known that phenolic acids in general are capable of reducing the formation and multiplicity of ACF (Borrelli et al., 2002). Thus, the presence of two polyphenol rings in RA could have conferred to its growth inhibitory effect. Oxidative stress produced by free radicals may initiate chain reactions that directs the damage to cell membranes through lipid peroxidation. Tumour cells acquire the ability to protect

themselves from lipid peroxidation process, thereby it increases cell proliferation in a short period. In addition, tumour cells get resistant to free radical attack, so lipid peroxidation may not be severe (Nakagami et al., 1999). Thus in our study the reduced levels of lipid peroxidation in the colon tissue could be attributed to the tumour burden. These results are consistent with our previous reports (Aranganathan and Nalini, 2009; Sreedharan et al., 2009) and also by others (Bartoli and Galeotti, 1979). Metabolism of DMH involves several xenobiotic metabolizing enzymes, which activates the procarcinogen. Elevated levels of CYP2E1 was reported in DMH induced colon cancer (Sohn et al., 1987). Thus, the inhibitions of CYP2E1 is potentially important in xenobiotic toxicity, because it could either attenuate or potentiate the toxicity of xenobiotics. The procarcinogen DMH induces CYP2E1 and thus the conversion of the ultimate carcinogen through the biotransformation, leading to the formation of AOM (DMH treated rats). Phenolic acids are known to be powerful tools to modulate carcinogen metabolism, particularly those in which CYP2E1 is involved (Krajka-Kuzniak et al., 2005). Being a phenolic acid RA prevents DMH induced activation of CYP2E1 as well as the conversion of chemicals into carcinogens. An increase in phase II detoxification enzymes might be considered to be beneficial, since this could enhance the excretion of carcinogens. Our results suggest that supplementation with RA during the initiation phase might not have enhanced the activity

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Fig. 9. Immunohistochemical expression of COX-2 in the colon of control and experimental rats. A and B: Colonic sections of control and controlþ RA showing standard COX2 expression. C: Colonic section of DMH treated rat showing strong COX-2 expression. D, E and F: Colonic sections of DMH treated rats supplemented with RA showing moderate to mild COX-2 expressions during the initiation, post initiation and entire period treatment regimens. (n ¼6).

of the detoxifying enzymes sufficiently to detoxify the ultimate carcinogen, DMH. However, supplementation with RA for the whole experimental period significantly enhanced the activity of the detoxification enzymes, rendering the easy elimination of the carcinogen, DMH and its metabolites. In this context, active compounds from plant sources are known to inhibit the cytochrome P-450 system responsible for converting carcinogenic agents into forms capable of covalent binding with DNA (Mandal et al., 1987).

Overall our results suggested that RA treatment might enhance the detoxification mechanism by modulating the phase I and phase II enzymes. β-glucuronidase deconjugates conjugated toxin and carcinogens glucuronides in the lumen and gut, it may lead to the formation and recirculation of ultimate carcinogen in the colon (Adlercreutz et al., 1976; Kuhn, 1998). Enhanced activity of β-glucuronidase was found in carcinogen alone exposed rats (group 3)

K. Venkatachalam et al. / European Journal of Pharmacology 791 (2016) 37–50

imply that occurrence of elevated levels of MAM deconjugation therefore the ultimate carcinogen again entering in to the enterohepatic recirculation. β -glucosidase hydrolysis plant glycosides in the gut, which can release toxic mutagenic and carcinogenic aglycones. Ohkami et al. (1995) reported that bacterial βglucosidase hydrolyzes DMH to its toxic metabolite, methylazoxymethanol leads to excess generation carcinogens from conjugated carcinogen. Administration of RA to tumour induced rats, reduces the activities of β-glucuronidase, β-glucosidase and βgalactosidase thereby increases the elevation of carcinogen through faeces. When there is any change in the amount and/or the composition of mucus may lead to inflammatory responses (Linden et al., 2008). Augmented degradation of mucin is noticed during the direct contact of the colonic tissue, which is transformed to neoplastic cells (Goldin and Gorbach, 1984). Decreased faecal mucinase activity was observed in carcinogen exposed RA supplemented rats. This could be attributed to the chemopreventive potential of RA. Nitroreductase reduces heterocyclic and aromatic nitro compounds, which are extensively used in the industries and medicine, to carcinogenic derivatives (Lee and Lee, 2001). The elevated levels of nitroreductase were observed in people consuming diet linked to an increased risk of colon cancer (Goldin and Gorbach, 1977). Sulphatase also involve in deconjugates, which degrades sulphated mucins. The levels of nitroreductase and sulphate were elevated in DMH treated rats parallel with increased tumour incidence. RA administration to DMH exposed rats brought back the levels thereby reduce the tumour incidence. Augmented expression of Bcl-2 prevent the flow of cytochrome C from mitochondria thereby prevented the apoptosis. In DMH alone treated rats we observed an elevated level of Bcl-2 and reduced levels of Bax, p53, caspase-3 and -9 proteins, which showed that the tumour cells might have acquired the ability to evade apoptosis (Jornot et al., 1997). Supplementation with RA to DMH exposed rats induced apoptosis, as evidenced by increased expressions of p53 and the release of cytochrome c from mitochondria that lead to the activation of caspase-3, a key protease in the execution of apoptosis. Regular supplementation with RA throughout the experimental period showed the activation of apoptosis in tumour cells by reduces Bc-2, increases Bax and the induction of p53 and caspase pathways, thereby preventing tumour cell proliferation and evasion of apoptosis. In this context, polyphenols can act as antioxidants as well as prooxidants depending on the tumour environment. Oxidation of polyphenols produces O2, H2O2 and a complex mixture of semiquinones and quinones, all of which are potentially cytotoxic (Cai and Jones, 1998; Tamarit et al., 1998). In DMH injected tumour bearing rats have different types of molecular alterations may result in impaired regulation of NF-κB. During this event, NF-κB loses its inducibility and becomes constitutively activated. This leads to deregulated expression of genes under NF-κB control. Since alterations in all these processes participate in development and progression of cancer, there is a clear link between NF-κB and carcinogenesis. So, NF-κB activation is the result of a multistep signalling pathway. On supplementation with RA the levels of p65 protein expression was reduced, which implies that RA inhibits the NF-κB mediated cell proliferation. Overall our results suggests that, RA prevents the formation and multiplicity of ACF and tumours, protects cells from the damages caused by DMH and its metabolites, spares the activities of antioxdiant enzymes and induces apoptosis in tumour cells by virtue of its ability to act as a chemopreventive agent.

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Acknowledgement The financial assistance offered by the Indian Council of Medical Research, New Delhi, India, in the form of Senior Research Fellowship (IRIS ID:2010-04190) to V. Karthikkumar is gratefully acknowledged.

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